Ion channels play an important role in a wide range of cellular and system physiology, including generation, processing and modulation of electrical signals in excitable cells and tissues. Detailed knowledge of ion channel function is essential for an understanding of normal and pathological physiology that will facilitate new treatments for a wide variety of diseases such as epilepsy, pain, and cardiovascular disease. In this proposal we focus on the small-conductance, calcium-activated potassium (SK) channel (KCa2, or KCNN2). SK channels are important for a wide range of physiological systems, and are involved in cerebellar ataxia, epilepsy and learning and memory. SK channels use the ubiquitous calcium-binding protein calmodulin to sense changes in intracellular calcium. The molecular mechanism by which calcium binding to constitutively-associated calmodulin opens the SK channel pore is poorly understood. Our goal is to develop an accurate, quantitative understanding of this process. A critical barrier to further progress on this important problem is the lack of methods for measuring ligand binding and channel activation simultaneously. We will combine electrophysiological measurements of channel activation with a novel spectroscopic method (conditional binding) for quantifying calcium binding to SK channels. Our conditional binding method uses energy transfer between luminescent lanthanide probe ions to assess the occupancy of two EF hand calcium binding sites within the same calmodulin molecule simultaneously. Extensive theoretical analysis of this phenomenon demonstrates that, by isolating the binding signals from a small subset of ligated configurations of the SK/CaM complex, conditional binding measurements are both necessary and sufficient for estimating microscopic, site-specific binding affinities and cooperative interactions. Preliminary binding measurements on free calmodulin and on SK/CaM complexes demonstrate the feasibility of our approach for developing quantitative models of SK channel function. The binding and gating measurements are performed simultaneously on functional SK channels in excised membrane patches, ensuring the relevance of the derived model parameters to the channels' in vivo behavior. Our conditional binding method can be applied to any molecular system containing paired EF-hand binding sites, the largest category of calcium binding proteins in biology. This enormous group includes the majority of calcium-modulated ion channels, as well as many other important effector proteins such as the calmodulin-dependent kinases and calcium pumps. Our studies will contribute to understanding the function of an important class of ion channel, and general principles of ion channel gating, calcium/calmodulin regulatory mechanisms, and allosteric control of protein function.